Since proteins are found in every membrane and aqueous compartment within cells and yet are primarily synthesized on cytoplasmic ribosomes, protein targeting and transport across or into lipid membranes is a fundamental process in all organisms. Many distinct types of translocation systems exist that allow large protein molecules to cross membranes without compromising the membranes' role as a permeability barrier to ions, metabolites, and macromolecules. While many protein transport systems translocate 'linearized polypeptides', the twin-arginine translocation (Tat) system transports fully-folded and assembled proteins without collapsing ion gradients. The absence of a functional Tat system in bacteria often leads to growth defects and occasionally death. The Tat machinery is also responsible for the export of proteins important for bacterial virulence in humans. Since animals, including humans, do not contain homologues of Tat machinery proteins, inhibitors of Tat transport could potentially find use as novel antibiotics. Our long-term goal is to decipher the mechanism of protein transport by the bacterial Tat machinery at a molecular level. In past research, numerous in vitro assays that enable biochemical and biophysical investigations of the Escherichia coli Tat transport mechanism have been developed. Recent work indicates that the Tat machinery catalyzes insertion of a signal peptide hairpin into the membrane in an energy-independent manner, and that full translocation of the C-terminal end of the signal peptide requires a proton motive force. These results establish that the signal peptide's binding interactions and its membrane translocation are critical for directly promoting mature domain transport. However, the structural and oligomeric nature of the translocation pore and how leakage is prevented remain major unsolved problems. Characterization of the translocation pore is particularly challenging because it disassembles in the absence of a proton motive force. We will examine structural and dynamic properties of the Tat machinery, and test hypotheses generated by our Hairpin-Hinge Model of transport. Our Specific Aims are: (1) to identify the signal peptide binding site on the TatBC receptor complex, and determine the arrangement of TatBC heterodimers in the TatBC oligomer; (2) to characterize translocon and cargo dynamics during Tat transport using real-time kinetic approaches; and (3) to identify the conformational and environmental changes that TatA experiences during transport. We will use crosslinking and real-time fluorescence approaches, including single molecule studies, to probe binding interactions and the dynamic transitions between important structural intermediates in the translocation cycle. This work will clearly establish the manner in which signal peptides interact with the Tat machinery, and the structural, dynamic, and oligomeric properties of the translocation pore. In total, this study will significantly advance our understanding of the Tat transport mechanism, which will in turn provide a sound framework for understanding bacterial growth and virulence, and for the development of antibiotics and biotechnological tools.